1. Field
The present technology is related to methods and compositions for real-time PCR and other nucleic acid detection, extension or amplification reactions.
2. Description of the Related Art
Numerous methods of amplification of a nucleic acid are known to those skilled in the art. In general, the amplification of a nucleic acid sequence includes creating one or more copies of the nucleic acid sequence or of a secondary nucleic acid sequence intended to be indicative of the presence of the first nucleic acid. Examples include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), loop-mediated isothermal amplification (LAMP), among others. In some technologies, a nucleic acid sequence indicative of the presence of a non-nucleic acid structure (e.g., a protein) is amplified, as in the proximity ligation assay (PLA) and other amplification methods.
Real-time and endpoint detection methods in these amplification reactions can involve the use of dyes that have a different fluorescent output in the presence of double-stranded deoxyribonucleic acids (DNA), the use of sequence-specific probes or the use of primers with attached detection mechanisms. Examples of primers with attached detection mechanisms include SCORPION™ primers (DxS Ltd., Manchester, UK) and AMPLIFLUOR® primers (Millipore, Billerica, Mass.).
SCORPION™ primers have a stem-loop nucleic acid sequence which binds specifically to itself through a scorpion-like orientation or mechanism. Because the stem-loop sequence is specific to the product formed by the primer extension, the design of scorpion primers requires three unique, conserved sequences. In some highly polymorphic organisms, finding even one conserved sequence is difficult, let alone finding three conserved sequences.
Hence, primers such as the AMPLIFLUOR® primer—which has a similar structure to SCORPION™ primers (i.e., a primer attached to a stem-loop structure) but without the requirement of a third conserved binding site—are in demand. However, the AMPLIFLUOR® detection method works by incorporating the hairpin structure into the amplified product, and therefore requires a conserved sequence sufficiently long to bind both the primer and the sequence from a stem-loop structure. A method that allows for more flexibility in designing detection primers for unique, conserved regions is therefore needed.